WO2013154429A1 - Multiplex reporter assays for monitoring multiple variables - Google Patents

Abstract

The disclosure relates to multiplex assays for monitoring multiple variables, such as the effects of a biological condition on a biological system. Reporter systems are provided for performing such assays.

Description

Title: Multiplex reporter assays for monitoring multiple variables

FIELD OF THE INVENTION

The disclosure relates to multiplex assays for monitoring multiple variables, such as the effects of a biological condition on a biological system. Reporter systems are provided for performing such assays.

BACKGROUND OF THE INVENTION

To study complex processes in systems biology efficiently, tools that give insight into multiple molecular and cellular interactions simultaneously and in real-time are needed^{1 2}. Several cellular reporter systems are in use to shed light on biological processes, many based on luciferase enzymes and their photon production upon luciferin substrate addition, including Firefly luciferase (Flue)³, Renilla luciferase (Rluc)⁴, and Gaussia luciferase (Glue)^{5 6}. These bioluminescent reporters have been used to monitor biological processes such as cellular proliferation and differentiation⁷, transcription factor activity⁸ and translational repression^{9 10}, and intercellular interactions¹¹, reviewed in¹².

Other commonly used reporters are based on the green fluorescent protein (GFP), the red fluorescent protein (RFP) and more recently, near infra-red fluorescent proteins^{13 14} which can be multiplexed together to monitor several processes simultaneously using spectral unmixing in conjunction with fluorescence molecular tomography¹⁵.

Monitoring several variables simultaneously has been challenging due to difficulties of detecting multiple reporter signals. There is a need for improved multiplex screening assays, in particular those suited for real-time studies. SUMMARY OF THE INVENTION

The disclosure provides improved multiplex screening assays for the detection of multiple reporter signals. In one aspect, the disclosure provides a method for monitoring an effect on a biological system comprising

providing said biological system with at least two reporters,

wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to a reporter;

exposing said biological system to a biological condition; and

determining the level of said reporters.

Preferably, the biological system is one or more cells, more preferably the cells are tumor cells.

Preferably, said methods further comprise distinguishing said reporter based on the epitope tag.

Preferably, said reporters are provided to said biological system in vitro.

Preferably, said biological system is implanted into an animal. Preferably said reporter level is determined in a bodily fluid, preferably in the blood, of said animal.

In one aspect, the disclosure provides reporter systems. These reporter systems are ideally suited for use in the methods disclosed herein. Reporter systems are provided comprising at least two reporters,

wherein each reporter comprises a marker, preferably a luminescent marker, linked to an epitope tag, and each epitope tag is unique to each reporter.

Preferably, the marker from each reporter is the same. Preferably, the marker is secreted, preferably wherein the marker is Gaussia luciferase. Preferably, wherein each reporter comprises a different transcription response element or a different promoter sequence.

Preferably, each reporter comprises a different protease cleavage site.

Preferably, each reporter comprises a different miRNA binding sequence or a different RNA splicing sequence.

Preferably, the epitope tag is selected from Flag, His, HA, AcV5, V5, Glu, Myc, Kt3, Aul, and E2, more preferably wherein said epitope tag is selected from Flag, His, HA, AcV5, V5, and Glu.

In preferred embodiments, the reporters are provided in one or more vectors. In a further aspect, nucleic acid encoding the reporter system disclosed herein is provided. In a further aspect, cells comprising said nucleic are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Gluctag multiplex assay development and validation in vitro, (a) Schematic of lentiviral vector constructs encoding the luciferase reporters, (b) Fluorescence microscopy analysis of a representative U87-FM-Gluctag-CFP cells (using GlucFiag reporter) showing mCherry levels (in red) and CFP expression (in blue; marker for transduction efficiency), (c) Immunostaining against the various tags in U87-FM cells expressing different Gluctag. (d) Glue activity in U87 cells expressing different Gluctag with respect to cell

proliferation over time and cell number. The dashed line represents Glucctri activity, (e-f) Gluctag immunobinding assay versus total Glue activity using serial dilutions of the conditioned medium from one day culture of either individual U87-FM cells expressing a single Gluctag or a mixture of cells expressing all ten different Gluctag (f). (g) Immunostaining for the ten different tags expression in the mixed population of U87-FM cell culture. Size bar (in b,c and g) = 100 μιη. (h) Immunostaining for the ten different tags in one cell line. The insert panels show staining of control parental cells. Percentages of positively stained cells are indicated, (i) Immunobinding assay of all ten Gluctag reprters in one cell line after TMZ treatment (black bars) compared to untreated controls (white bars), (j) CFP fluorescence microscopy of cells in (h-i) with or without 600 micromolar TMZ.

Figure 2. Gluctag multiplex assay in vivo, (a) U87-FM cells expressing different Gluctag were implanted subcutaneoulsly in different mice and tumor growth was monitored overtime. Shown is a representative Flue bioluminescence imaging of the U87-FM-Gluctag-CFP cell lines using GlucFiag reporter, (b), Quantitation of the different U87-FM tumors expressing different Gluctag reporter; Flue activity (black), calliper measurement (green), total Gluctag activity in the blood (red), and bound Gluctag activity after immunobinding (blue), (c-d) U87-FM cells expressing different Gluctag were mixed in equal ratio and injected subcutaneously in the same mouse (n=5). Tumor growth was monitored using Flue imaging and total Glue blood assay as in (a-b). Individual cells subpopulation within the same tumor were monitored using the multiplex Gluctag immunobinding assay, (e) Immunostaining for the expression of the ten different tags in the same U87-FM-Gluctag-CFP tumor. Black arrows indicate positive staining for cells expressing the corresponding Gluctag. Size bar = 100 μιη. (f-g) Mixed population of U87-FM cells expressing all ten different tags were implanted intracranially in the brain of nude mice (n=5). Tumor growth was monitored with Flue imaging and Glue total blood assay, and different tumor cells subpopulation was monitored using the multiplex Gluctag immunobinding as in (c-d). Flue bioluminescence imaging from a representative mouse is showing in (f). Data shown in (b,d, abd g) as average relative light units (RLU; n=5) ± standard deviation.

Figure 3. CSCW-Gluc-IRES-CFP vector

Figure 4. GlucTag cloning strategy. The Glue gene is amplified from the parental vector using primers that amplify Glue without the tata-box and adding an Xbal and Xhol restriction site downstream. This modified Glue gene then replaces the original Glue gene in the parental vector to construct the Glucmodified vector. Then, the vector is restricted with Xbal and Xhol to insert an epitope tag of choice. This GlucTag is the final construct.

Figure 5. GlucTag Immunostaining in mouse brains

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

The present invention is based on a reporter system that allows monitoring of multiple variables within a biological system. We demonstrate herein that the system is both sensitive and specific enough to permit real time monitoring of, e.g., mixed populations of tumor cells both in vitro and in vivo (see Figures 1 and 2).

The reporters described herein may be used in a variety of different

applications and are particularly useful for monitoring multiple variables within a biological system. Previous multiplex assays were based on

monitoring each variable using a different luminescent marker. The ability to distinguish markers emitting light at different wavelengths requires expensive equipment and increases the costs of such assays. In contrast, the present invention provides a reporter system comprising both a marker and an epitope tag. Preferably, each reporter has the same marker and the epitope tag is used to discriminate between reporters. The present reporter system can be used for continuous reporter measurement both in vitro and in vivo. Accordingly, the present disclosure provides a reporter system comprising at least two reporters, wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to each reporter. Preferably the reporter system comprises at least three, at least four, or at least five reporters, wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to each reporter. Reporter systems comprising at least 10 different reporters can distinguish biologically relevant differences (see Figures 1 and 2).

As used herein, a reporter refers to a protein comprising a detectable marker and an epitope tag.

Preferred markers of the reporter system include luminescent and colorimetric markers. Examples of colorimetric labels include compounds which use a chromogenic substrate to produce a color, such as beta-galactosidase, beta- glucouronidase, horseradish peroxidase, and alkaline phosphatase.

As used herein, luminescence refers to the emission of light by energy other than heat and includes bioluminescence, fluorescence, and phosphorescence. Luminescent markers include, e.g., bioluminescent markers, fluorescent markers, and phosphorescent markers. Preferably the luminescent marker is a peptide or protein molecule.

Suitable fluorescent protein markers include Y66H, Y66F, EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, ECFP, CyPet, Y66W, mKeima- Red, TagCFP, AmCyanl, mTFPl, S65A, Midoriishi Cyan, Wild Type GFP, S65C, TurboGFP, TagGFP, S65L, Emerald, S65T (Invitrogen), EGFP

(Clontech), Azami Green (MBL), ZsGreenl (Clontech), TagYFP (Evrogen), EYFP (Clontech), Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellowl (Clontech), Kusabira Orange (MBL), mOrange, mKO, TurboRFP (Evrogen), tdTomato, TagRFP (Evrogen), DsRed (Clontech), DsRed2 (Clontech), mStrawberry, TurboFP602 (Evrogen), AsRed2 (Clontech), mRFPl, J-Red, mCherry, HcRedl (Clontech), Katusha, Kate (Evrogen), TurboFP635

(Evrogen), mPlum, and mRaspberry. Preferably, the marker is a bioluminescent marker. Bioluminescence refers to the emission of light from a living organism and is a common trait in deep-sea marine organisms. It also occurs, e.g., in arthropods, fungi, and

microorganisms. Bioluminescence is the product of a reaction catalyzed by an enzyme, i.e., luciferase. As used herein, bioluminescence also refers to the emission of light resulting from a reaction catalyzed by a luciferase.

Luciferases are proteins which react with a suitable substrate to produce light as one of the reaction products. Luciferases catalyze the oxygen oxidation of an organic molecule, i.e., a luciferin (such as aldehydes, benzothiazoles, imidazolopyrazines, tetrapyrroles and flavins). Luciferases that use

coelenterazine (an imidazoloyrazine derivative) as a substrate to produce luminescence include luciferases from the species Renilla, Gaussia, Metridia and Obelia. The amount of light produced by a bioluminescent reaction can be measured and used to determine the presence of or amount of luciferase in a sample. The term "luciferase" refers to a naturally occurring or mutant luciferase

Preferably, the marker protein is secreted. This permits the detection of the reporter in, e.g., conditioned cell culture medium in vitro or in a bodily fluid such as blood. Secretion of the marker protein can be achieved by using a naturally secreted marker protein or by engineering a signal peptide into the marker sequence. As used herein, a signal peptide is a sequence which is present as an N-terminal sequence on the precursor form of an extracellular protein. The function of the signal peptide is to allow the heterologous protein to be secreted to enter the endoplasmatic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the host organism producing the protein.

Secreted markers include glucoamylase::green fluorescent protein fusion, (Gordon et al. Microbiology 2000 2:415-426), green fluorescent protein with ecdysteroid UDP-glucosyltransferase signal peptide (Laukkanen et al.,

Biochem and Biophy Res Comm 1996 3:755-761), Gaussia luciferase, and Metridium luciferase. Preferably the marker is Gaussia or Metridium luciferase.

In preferred embodiments, the marker is Gaussia luciferase (Glue). Glue is highly sensitive, naturally secreted, and can be detected in the conditioned medium of cells in culture as well as in the blood of mice ex vivo, allowing realtime monitoring of cellular processes^{5 6}. An exemplary sequence of Glue is provided herein. Glue also includes variants which have been codon optimized for use in prokaryotes or eukaryotes (see, e.g., Wille et al. Applied and

Environmental Microbiology 2012 78:250-257). Glue also includes variants which have been modified, e.g., for prolonged bioluminescence, increased bioluminescence intensity, or bioluminescence stability (e.g., US Patent Application No. 20120034672, which is hereby incorporated by reference)

The marker is linked to an epitope tag. As used herein, an epitope tag is an amino acid sequence that may be specifically bound by another moiety, usually another polypeptide, most usually an antibody. An epitope tag also includes any epitope that is experimentally determined to raise a monoclonal response in an animal that results in a high-affinity antibody for a defined peptide epitope.

An epitope tag is a peptide sequence heterologous to the marker, i.e., the reporter is a fusion protein of a marker and an epitope tag.

Suitable epitope tags include Flag, His, HA, AcV5, V5, Glu, Myc, Kt3 (Martin et al., Science, 255: 192-194 (1992), Aul, E2, glutathione S transferase (GST) and maltose binding protein (MBP), polyoma virus T antigen epitope, and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990). Although larger proteins may be used, e.g., GST and MBP, smaller epitopes are preferred, for example epitopes between 5 to 50 amino acids in length. A skilled person would recognize that other epitope tags are also suitable in the present invention.

In preferred aspects of the disclosure, the epitope tag is selected from Flag, His, HA, AcV5, V5, Glu, Myc, Kt3, Aul, and E2. More preferably, the tag is selected from Flag, His, HA, AcV5, V5, and Glu. These tags showed the highest efficiency in terms of antibody binding in present examples. However, a skilled person recognizes that other tags and/or their respective antibody or tag- binding moieties tags could be optimized to achieve higher sensitivity and are thus encompassed by the invention.

In preferred aspects of the disclosure, the nucleic acid encoding each reporter comprises a transcription factor response element (TRE). A TRE is the nucleic acid sequence that a transcription factor binds to. In preferred embodiments, the TRE is selected from the binding sequence for P53, NF-kB, Hif-la, E2F, Creb, SP-1, AP-1, Stat3, Sox2, Klf4, Nanog, c-Myc, Elk, and Oct4. However, it is clear to a skilled person that any TRE is encompassed by the present invention. These transcription factor binding sequences are well-known in the art. Binding sequences can also be predicted by a number of computational tools, such as TFSEARCH, TRANFAC, Matlnspector (Cartharius K (2005) Bioinformatics 21, 2933-42). Binding sequences can also be confirmed or determined experimentally using such protocols as DNase I Hypersensitivity, EMSA, and ChlP. Further details for both computational and experimental determination of TREs may be found in Elnitski et al. Genome Research 2006 16: 1455, the contents of which are hereby incorporated by reference in their entirety. Expression of the reporter provides an indication of the effect of a variable on a TRE. Preferably, each reporter comprises a different TRE.

In exemplary embodiments, TRE sequences may be flanked with restriction sites for cloning. Suitable primer sequences are disclosed herein, although it is understood that different restriction sequences may be used or in some cases the addition of restriction sites may not be necessary.

In preferred aspects of the disclosure, the nucleic acid encoding each reporter comprises a promoter sequence. A promoter sequence is a nucleic acid sequence capable of initiating transcription. Promoters may be constitutive wherein the transcription level is constant and unaffected by modulators of promoter activity, e.g., CMV. Promoters may be inducible in that promoter activity is capable of being increased or decreased, for example as measured by the presence or quantitation of transcripts or translation products. Promoters may also be cell specific wherein the promoter is active only in particular cell types. Expression of the reporter provides an indication of the effect of a variable on the promoter. In some embodiments, a viral promoter is operably linked to the reporter. Upon infection of the cell by a virus, the viral promoter is activated and detection of the specific reporter provides a measure of viral infection. Preferably, each reporter comprises a different promoter. In preferred aspects of the disclosure, each reporter comprises a protease cleavage site. In one embodiment, a protease cleavage site is inserted in between the marker and the epitope tag. Suitable cleavage sites include those for caspase proteases; viral proteases, e.g., HIV protease; proteases of bacterial toxins (e.g, botulinum toxin); proteases that process cellular proteins, e.g., secretase processing of beta-amyloid, proteases regulating cell adhesion (e.g., metalloproteases associated with extracellular matrix); proteases involved in blood coagulation, inflammation and would healing; and tumor cell associated proteases. In some embodiments of the disclosure, the protease cleavage site is located between the marker and the epitope or within the marker itself.

Cleavage at the site would therefore result in a decrease in signal. Preferably, each reporter comprises a different protease cleavage site.

In preferred aspects of the disclosure, the nucleic acid encoding each reporter comprises an miRNA binding sequence. The binding of an miRNA to its respective binding sequence usually leads to translational repression or target degradation. Expression of the reporter, or lack thereof, provides an indication of the effect of a variable on the miRNA binding and silencing process.

Preferably, each reporter comprises a different miRNA binding sequence.

In preferred aspects of the disclosure, the nucleic acid encoding each reporter comprises an RNA splicing sequence. The RNA splicing sequence may be inserted either in the marker or epitope tag or between the two. Preferably, splicing leads to a fusion protein of the marker and epitope tag. Such reporter systems are useful, e.g., in monitoring the efficiency of a splicing sequence or splicing machinery in different cells types or under different biological conditions. Preferably, each reporter comprises a different RNA splicing sequence. As is clear to a skilled person, the reporter systems disclosed herein may be used in additional applications and may also include additional sequences.

Preferably, the reporters are provided as fusion proteins, nucleic acids encoding said fusion proteins, or as vectors comprising said nucleic acids.

Preferably, the reporters are provided such the epitope tag is fused in frame at the N- or C-terminus of the marker sequence. Methods for producing fusion proteins are well-known in the art. Accordingly, nucleic acids are provided comprising a nucleic acid sequence encoding a marker as disclosed herein and a nucleic acid sequence encoding an epitope tag as disclosed herein. The nucleic acids described herein can be prepared using standard recombinant DNA techniques described in, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.

The nucleic acids will usually also containing sequences linking the marker and epitope tag sequences, such as enzyme restriction sites which can be used to facilitate cloning. Additional linkers, such as sequences encoding Gly and/or Ser residues may be also be present. In a reporter system, each reporter may be provided on a separate nucleic acid or on the same nucleic acid, e.g., a vector may be provided comprising two or more reporters.

Said nucleic acids may be operably linked to additional sequences such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In a preferred embodiment, the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element. Preferably, the nucleic acid also comprises a sequence encoding a signal peptide. Signal peptides target the protein to the secretory pathway and are well-known in the art. The signal peptide may be an endogenous or exogenous sequence. Preferably, the nucleic acids are provided in vectors. Accordingly, vectors are provided comprising a nucleic acid sequence encoding a marker as disclosed herein and a nucleic acid sequence encoding an epitope tag as disclosed herein. Preferably, the vector also comprises one or more additional sequences as described above, such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop

sequences, and enhancer or activator sequences.

A "vector" is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors. Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

The nucleic acid sequences may also be provided in a delivery complex, e.g., in liposomes, electrically charged lipids (cytofectins), and biopolymers. Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided. The method of introduction is largely dictated by the targeted cell type include, e.g., CaPO₄ precipitation, liposome fusion, lipofectin,

electroporation, dextran-mediated transfectionpolybrene mediated

transfection, protoplast fusion, viral infection, encapsulation of the

polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. The nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).

The reporters as described herein may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a reporter polypeptide. Appropriate host cells include yeast, bacteria,

archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc. Preferably, said polypeptides are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. Suitable cell types include tumor cells (such as U87 human glioma cells), Jurkat T cells, NIH3T3 cells, CHO, and Cos cells. Preferably, said polypeptides are expressed in bacterial systems. Bacterial expression systems are well known in the art.

The reporters described herein are particularly useful for monitoring multiple variables, preferably in response to a biological condition. Accordingly, methods are provided for monitoring the effect on a biological system comprising providing said biological system with at least two reporters as described herein, exposing said system to a biological condition; and

determining the level of said reporters. Preferably, at least three or at least four reporters are provided.

As used herein, a biological system includes a biological system or a bodily fluid. A biological system includes a cell, a tissue sample, cell extract, a bodily fluid, as well as a viral particle. Any type of cell may be used, such as animal (e.g., mammalian, avian, insect), plant, fungal (e.g., yeast), bacterial cell, or any type of viral infected cell. A bodily fluid, as described herein, includes blood, serum, plasma, amniotic fluid, brain/spinal cord fluid, liquor,

cerebrospinal fluid, sputum, throat and pharynx secretions and other mucous membrane secretions, synovial fluids, ascites, tear fluid, lymph fluid and urine.

A "biological condition" includes in vivo or in vitro growth conditions, administration of a test compound (such as a small molecule, nucleic acid, or protein), infection (e.g., by a virus, parasite, micro-organism) and stress conditions (e.g., heating, cooling, toxins, varying osmotic conditions).

Preferably, the biological system is a cell. Suitable cell types include, but are not limited to, tumor cells of all types (e.g., glioma, melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells, osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, and Cos cells. The reporter system may be provided to the biological system in vitro, in vivo, or ex vivo. The reporter system may be provided as nucleic acid (e.g., nucleic acid can be provided to cell extracts or transformed into cells or tissues) or as expressed proteins. The biological system may then be exposed to a biological condition, which may include implanting the biological system into an organism, preferably a mammal.

The reporter system described herein may be provided to the same biological system (e.g., multiple reporter molecules provided to a single cell) or to different biological system (e.g., a first reporter provided to a first cell and a second reporter provided to a second cell; or a first and second reporter provided to a first cell and a third and fourth reporter provided to a second cell). The second biological system may be a different type of biological system from the first biological system (e.g., the first cell may be a wild-type cell and the second cell may have a mutatation) or may have been subjected to a different condition or treatment, e.g., exposure to a compound or infection.

When provided to different biological systems, the reporter system has the advantage that the effects between different biological systems (e.g., different cells) can be monitored. In an exemplary embodiment, cells are exposed to a biological condition and the level of the reporter is then determined in the first cell and the respective level is determined in the second cell. The respective levels of the reporters provides an indication of an effect on the cells.

The effect on the cells is preferably due to the effect of a biological condition on a biological system. Said condition may result, e.g., in the increase in RNA transcription, protein translation, cell proliferation, etc., which would generally lead to an increase in the level of a reporter. In a preferred embodiment, methods are provided for monitoring an effect on a cell comprising providing said cell with at least two reporters as described herein, exposing said cell to a biological condition; and determining the level of said reporters produced by said cell. If the cells are allowed to proliferate, then the reporter levels can also be used as an indication of proliferation, or a lack thereof (or alternatively cell death).

In some embodiments, the epitope tag is used to immobilize the reporters on a surface (e.g., well-plate, bead, microsphere, or chip) before marker detection. Preferably, reporters are separated from each other using immunobinding. The surface comprises an epitope tag binding molecule, such as an antibody specific for an epitope tag, which allows the surface, via the binding molecule, to bind the epitope tag, i.e., the reporters are captured on the surface. The epitope tag binding molecules are specific for the epitope tags used in the reporter system.

Preferably, the methods disclosed herein comprise the steps of providing a biological system with at least two reporters as disclosed herein, exposing said biological system to a biological condition; contacting said system with one or more surfaces, and determining the level of marker bound to each solid substrate.

Epitope tag binding molecules, e.g., an antibody, can be linked to a surface by any method known in the art. Suitable surfaces include polymers, nylon, microarrays such as protein chips, the well of an assay plate, a filter, a membrane, a chromatographic resin, a bead, or microsphere. Preferably, each surface with a particular epitope tag binding molecule is separated such that the identity of each reporter can be distinguished based on the epitope tag (and its binding properties). The marker is used to quantitate the amount of reporter. For example, each well of an assay plate or each spot on a chip or filter may have a different epitope tag binding molecule. In a preferred embodiment, the reporter system described herein is provided to cells (either the same or different cells as described herein) and said cells are then introduced into an organism, preferably an animal such as, e.g., a mouse, rat, rabbit, or human. Alternatively, said reporter system may be provided to cells or tissues within an organism. For example, one or more viral vectors encoding a reporter system described herein may be provided to a tumor within an individual. Bodily fluid for said individual may be collected in order to detect the level of the respective reporters. Preferably, the body fluid is blood, plasma or serum.

The reporter system may also comprise additional elements such as a transcription response element, promoter sequence, a protease cleavage site, miRNA binding sequence or RNA splicing sequence. These additional elements may be the same or different between the reporters. Preferably, the elements are the same when the reporter system is provided to different cells.

Preferably, the elements are different between each reporter when the reporter system is provided to the same cells.

The detection of the reporters as described herein may be carried out by any of the well-known methods known in the art. Fluorescent, luminescent and colorimetric labels and methods of detecting and measuring quantities with them are well-known and readily understood by those skilled in the art.

For the detection of luciferase activity, buffering agents such as Tricine, HEPPS, HEPES, MOPS, Tris, glycylglycine, or a phosphate salt may be present to maintain pH and ionic strength; a proteinaceous material, such as a mammalian serum albumin (preferably bovine serum albumin) or lactalbumin or an ovalbumin, that enhances the activity of luciferases in the luciferase- luciferin reaction, may be present; EDTA or CDTA

(cyclohexylenediaminetetraacetate) or the hke, may be present, to suppress the activity of metal-containing proteases or phosphatases that might be present in systems (e.g., cells) from which luciferase to be assayed is extracted and that could adversely affect the luciferase or the ATP. Glycerol or ethylene glycol, which stabilize luciferases, might be present. An exemplary assay is provided in the examples. When using a coelenterazine type luciferase (for example Glue or Mluc), the conditions described in US Application No. 20110256564 and hereby incorporated by reference may be used.

As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

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All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

EXAMPLES

We constructed a library of lentiviral vectors consisting of the Glue cDNA fused to ten different epitope tags at its C-terminus, under the constitutively active cytomegalovirus (CMV) promoter resulting in GlucFiag, GlucHis, GIUCHA, G1UCACV5, Glucv5, GIUCGIU, GlucMyc, GlucKt3, GlucAui, G1UCE2, and the control reporter construct without tag, Glucctri. This lentiviral vector also expresses cerulean fluorescent protein (CFP) separated from Gluctag by an internal ribosomal entry site (IRES) element as a marker for transduction efficiency (Fig. la and Online Methods section). We employed human U87 glioblastoma cells stably expressing Flue and the mCherry fluorescent protein⁶ (U87-FM) for validation of the Gluctag multiplex system. These cells were transduced with each of the lentivirus vector to produce ten different cell lines, each stably expressing a different Gluctag and CFP (U87-FM-Gluc_tag-CFP). Fluorescence microscopic analysis for mCherry and CFP showed that these cells were nearly 100% transduced with these vectors (Fig. lb). We first confirmed that all of the ten individual U87-FM-Gluctag-CFP cells expressed the corresponding tags by immunostaining using specific antibodies (Fig. lc). Cells expressing Gluc_ctri did not show staining with any of the tag-antibodies (data not shown). Next, we determined the effect of these tags on Glue by measuring its activity at different time points in an aliquot of conditioned medium from U87-FM cells expressing each Gluctag using the Glue substrate coelenterazine. We

demonstrated that fusion of epitope tags to Glue does not dramatically affect the expression, secretion or activity of Glue (Fig. Id). We then used the conditioned media containing the different Gluctag constructs to optimize antibody-tag immunobinding in a 96-wells plate format. As described in the Online Methods section, 96-wells plates were first coated with monoclonal antibodies against the different tags and incubated with different amounts of conditioned medium allowing Gluctag immunobinding. Subsequently, the wells were washed to remove unbound Gluctag and subjected to Glue assay by direct addition of coelenterazine to the wells and measuring photon counts using a plate luminometer. As a control, the same amount of conditioned medium was assayed directly for Glue activity (total Glue). We determined that six of the ten individual Gluctag constructs, i.e. GlucFiag, GlucHis, GIUCHA, G1UCACV5, G1UCV5, GIUCGI_U, reported with high-efficiency after immunobinding in the antibody- coated wells, as compared to total Gluctag activity (Fig. le). The GlucMyc, GlucKt3, GlucAui, and G1UCE2 reporters showed lower activity upon

immunobinding that was attributed to suboptimal antibody-tag interactions. In order to demonstrate the use of the Gluctag system for multiplex applications, we mixed equal numbers of the ten different U87-FM-Gluctag- CFP cells and plated them in a single well. Twenty-four hours later, we measured the total Glue level as above in an aliquot of the conditioned medium. Further, of the amount of the individual Gluctag in the medium was analyzed by immunobinding on wells coated with different antibodies directed against each of the individual tag. We were able to monitor in real-time the growth of the individual U87-FM-Gluctag-CFP cell population in the mixed cultures (Fig. If). Immunostaining confirmed the expression of each tag in the corresponding cells in the mixed culture (Fig. lg).

In order to determine the in vivo applicability of the Gluctag reporter assay, we injected the ten individual U87-FM cells expressing either one of the Gluctag or Glucctri reporters subcutaneously in different nude mice for singleplex monitoring. In another set, we injected mice with PBS as a negative control (12 groups; n=3/group). Tumor growth was monitored over time by calliper measurement, and Flue bioluminescence imaging (Fig. 2a,b). Blood was collected from mice at different time points and 5 μΐ (optimum amount for Glue blood assay¹⁶) was assayed for Glue activity (total Glue). At the same time, 50 μΐ of blood was analysed for Glue activity after immunobinding on wells coated with the corresponding tag antibody (Fig. 2b). All of our tags (except G1UCE2) allowed blood monitoring of tumor growth over time in a singleplex assay. We detected tumor growth as measured with a calliper, with Flue imaging and Glue blood assay and compared these methods with the signals of the ten different Gluctag reporters after immunobinding (Fig. 2b). The Gluctag reporters GlucFiag, GlucHis, GIUCHA, G1UCACV5, Glucv5, GIUCGIU were more sensitive in detecting tumor growth than others, probably due to a higher antibody to tag affinity. To demonstrate the use of the Gluctag system for multiplex

applications we mixed equal numbers of U87-FM cells expressing ten different Gluctag and implanted the heterogeneous cell pool subcutaneously in nude mice (n=5). We monitored tumor growth by Flue bioluminescence imaging and total Glue blood assay (Fig. 2c,d). Importantly, we were able to efficiently monitor the growth of the individual subpopulation of the U87-FM cells expressing the different high-efficiency Gluctag reporters (GlucFiag, GlucHis, GIUCHA, G1UCACV5, Glucv5, GIUCGI_U) in the same tumor over time (Fig. 2d). The other tags were not sensitive enough in detecting their corresponding subpopulation of tumor cells at this earlier tumor stage. We confirmed the expression of all tags (except the low-efficiency GIUCAUI) in the same tumor by immunostaining (Fig. 2e). To determine the applicability of the Gluctag multiplex system in deep tissues, the mixture of the ten different U87-FM-Gluctag-CFP cells were implanted orthotopically in the brain of nude mice (n=5). Tumor growth was monitored by Flue bioluminescence imaging and total Glue blood assay (Fig. 2f,g). Again, we were able to monitor the intracranial growth of the six individual

subpopulations of U87-FM cells expressing the high-efficiency Gluctag reporters in the same brain tumor, by blood sampling and Gluctag multiplex

immunobinding assay over time (Fig. 2g).

In conclusion, we have developed a Gluctag reporter system for real-time multiplex application and demonstrated its ability in monitoring individual cell populations in a mixed cell cultures in vitro, and in a single tumor in vivo both in subcutaneous model and in deeper tissues such as the brain. The individual epitope tags could also be employed to localize expression by immunostaining. Although we have used here ten different Gluctag reporters, some of which showed low efficiency in term of antibody binding, this tag library could be extended to virtually hundred different tags allowing the possibility of high-throuhput multiplexing applications. In addition, antibody binding of the low-efficiency tags in microtiter wells could be further optimized to achieve higher sensitivity since these different tags (except for GIUCAUI) were readily detected, by immunostaining. Real time monitoring of individual cells in a heterogenous mixture may allow, for instance, multiplexed RNAi screening or measurement of drug responses of multiple cell populations in parallel. The Gluctag multiplex system could be extended to monitor different variables in a single cell type. For instance, engineering each individual Gluctag under the control of a different transcription response element for multiplex transcription factor activity measurement; constructing miRNA-binding sequences in the 3'-UTR of the Gluctag constructs for multiplex miRNA activity monitoring; or by inserting different protease cleavage sites into the Gluctag gene for multiplex protease activity measurement. This reporter assay provides a valuable tool to study complex processes with different variables in systems biology.

Supplemental Methods

Cells. For in vitro and in vivo validation, we used the glioblastoma cell line U87, stably co-expressing Firefly luciferase and mCherry fluorescent protein. Cell lines were maintained in DMEM high glucose complemented with sodium pyruvate, stable glutamine, 10% FBS and pen/strep (all PAA), incubated under standard cell culture conditions of 37°C and 5% CO2.

Lentivirus vector construction and transduction. First, we amplified Glue by PCR using Accuprime Pfx DNA polymerase (Life Technologies) from CSCW- Gluc-IRES-CFP⁶ with specific primers which incorporates an Nhel site upstream (5' primer sequence) and Xbal site and Xhol site downstream (3' primer sequence) of the Glue cDNA. The 3' primer also contained a stop-codon after the Xhol site. The Glue cDNA, now containing a unique Xbal site, was ligated back into the CSCW-Gluc-IRES-CFP vector with T4 DNA ligase (Life Technologies) after removing of the wild-type Glue using Nhel and Xhol (Life Technologies). Plasmid DNA was transformed in XL- 10 Gold ultracompetent cells (Agilent Technologies), grown overnight on LB agar containing 50 μg/ml Ampicillin. We picked colonies to grow overnight and isolated DNA using a DNA plasmid mini kit (Qiagen) and verified successful transformation by Xbal restriction analysis. The Glue construct was then digested with Xbal and Xhol to insert an epitope tag. The epitope tags were designed with a Xbal site upstream, a stop codon and Xhol site downstream. A total of 20 μΜ of both single strand oligonucleotides of the acquired tag DNA (Life Technologies, see Table Si for sequences) were annealed in annealing buffer (100 mM Tris-HCl pH 7.5, 1 M NaCl and 10 mM EDTA) by heating to 65°C for 10 minutes and slow cooling down to room temperature. The epitope tag was then inserted into the vector using T4 DNA ligase and transformed in XL- 10 Gold ultracompetent cells. Bacteria were cultured and DNA was isolated. We verified the Gluctag constructs by sequencing using BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies). The Gluctag construct was then co-transfected with a third generation lentiviral packaging mix (pMDLg/pRRE, pRSV-Rev and pMD2.G) in HEK293T cells using Lipofectamine 2000 (Life Technologies). Virus was harvested 2 and 3 days after transfection and cell debris were spun down for 5 minutes at 1,000 x g. U87 cells were transduced overnight with lentivirus using a multiplicity of infection of 100 transducing units per cell in the presence of 8 μg/ml polybrene in standard culture conditions.

Fluorescence microscopy. Successful transduction was verified by visualising CFP (co-expressed with the Gluctag construct) and mCherry fluorescent protein (co-expressed with Flue) using fluorescent microscopy (Leica

Immunostaining and antibodies. For in vitro immunostaining, cells were fixed in 3.7% formaldehyde for 20 minutes, washed with PBS and permeabilized with PBS containing 0.1% Triton X- 100. Cells were then washed 3x in PBS containing 5% FBS and blocked with PBS containing 5% FBS. Primary mouse- anti-tag monoclonal antibody (10 μg/ml in 100 μΐ of PBS, Table Si) was added and incubated for 1 hour at room temperature. Cells were washed again and incubated with 1: 100 goat-anti-mouse horse radish peroxidase (HRP) conjugate (Dako) for 1 hour. After washing, cells were stained with DAB (Life Technologies). For mouse tissue staining, tumors were removed and embedded in paraffin. Microtome sections of 5 micron on glass slides were deparaffinised in xylene and rehydrated in ethanol series of 100, 96, and 70% ethanol.

Endogenous peroxide was blocked with 0.3% H2O2 in methanol for 30 minutes. After rinsing with water, antigens were retrieved with citrate buffer (pH 6) with 0.05% Tween 20 using a microwave (5 minutes 100%, 10 minutes 50% power). After slowly cooling, tissues were washed 3x with PBS and incubated with primary antibody (10 μg/ml), for one hour at room temperature. After washing again, tissues were incubated with envision anti-mouse and DAB stained as above (both Dako). Tissue sections were dehydrated with ethanol series as before and fixed in xylene. Cells and sections were imaged and photographed by light microscopy (Leica).

In vitro Glue activity assay. For Gluctag activity over time, 50,000 cells were plated in a 24 well plate and incubated overnight. 10 μΐ of conditioned medium were harvested from cells and Glue activity was measured by adding 50 μΐ (5 μg/ml) coelenterazine (Nanolight Technologies) in PBS and 0.1% Triton X-100). Before addition to the sample, the substrate was incubated at room

temperature for 30 minutes for stabilization. Photon count was determined over 10 seconds in a luminometer (Berthold Technologies). For the

measurement of Gluctag activity with respect to cell number, indicated numbers of cells were plated in a 24- well plate and incubated overnight.

Gluctag activity in medium was determined as described above. Gluctag immunobinding assay. White goat-anti-mouse-coated 96-well plates

(Thermo Scientific) were washed 3x with PBS containing 0.05% Tween 20 and incubated with 50 μΐ (10 μg/ml) of each of the mouse monoclonal antibody directed against the specific epitope tags (Table Si). Incubation time was 2 hours while centrifuging at 500 x g at 4 °C. Wells were washed and blocked 3 times with PBS containing 5% FBS and 0.05% Tween 20 on a plate shaker at 65-75 rpm. 30 μΐ of Gluctag conditioned medium or Gluctag mouse blood were added to the well and incubated for 2 hours at room temperature on a microplate shaker at 65-75 rpm. Wells were washed 5x for 5 minutes on a plate shaker at 65-75 rpm. A total of 50 μΐ Glue substrate (5 μg/ml

coelenterazine in PBS and 0.1% Triton X-100) was added to the well and photon count was determined in a microplate luminometer (Tecan) for 0.1 second per well. For the comparison of the bound Glue to the total Glue activity, aliquot of conditioned medium or 5 μΐ mouse blood was transferred to a white 96-wells microplate and assayed using 50 μΐ (5 μg/ml) coelenterazine in PBS and 0.1% Triton X-100 and a luminometer as above.

In vivo experiments. All in vivo experiments were subject to ethical committee approval and are conform VU university medical center and national regulations. For subcutaneous tumor xenograft model, 6 weeks old athymic Nude-Foxnl^nu mice (Harlan) were implanted with 5 x 10⁵ U87-FM-Gluc_tag-CFP cells in 50 μΐ DMEM and 50 μΐ Matrigel (BD Biosciences). For intracranial tumor model, a stereotactic frame (Harvard Biosciences) was used to inject cells vertically into the right hemisphere. Systemic anaesthesia was induced by subcutaneous injection of Temgesic (0.1 mg/kg). The mice were further anesthetized with oxygen containing 2.5% isofluorane. After removing the skin on the skull, drops of lidocaine (5 mg/ml in PBS) were administered to the incision. Coordinates used for injection are X = 0.5 mm, Y = 2 mm, Z = -2 mm from Bregma. A small drill was used to drill a hole into the skull. A total of 2 x 10⁵ cells in 3 μΐ of DMEM were injected vertically. On indicated days after injection, tumor size was monitored using a calliper (for subcutaneous) and bioluminescence in vivo Flue imaging, by injecting D-luciferin (100 mg/kg) intraperitoneally. Imaging was performed with an IVIS CCD camera and analyzed with Living Image software (Cahper Life Sciences). We collected 250 μΐ mouse blood aliquots from the tail vein in Microvette CB300 EDTA capillary tubes (Sarstedt) for ex vivo whole Glue measurements and Glue

immunobinding assays. Sequences

CMV-Gluc sequence: restriction sites are indicated in bold. [BamHI] [CMV promoter]

GGATCCCCCGGGCTGCAGGAATTCGATTAATAGTAATCAATTACGGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAA TGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAA TGAC GTATGTTC C C AT AGTAAC GC C AAT AGGGACTTTC C ATTGAC GTC AAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTA CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTA CATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC AC C C C ATTGAC GTC AATGGGAGTTTGTTTTGGC AC C AAAATC AAC GGGAC TTTC C AAAATGTC GTAAC AACTC C GC C C C ATTGAC GCAAATGGGC GGT AG GCGTGTACGGTGGGAGGTCTA

[Nhel] [Gaussia Luciferase] TATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCATGGGAGTC AAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCAC CGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGA CCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCC GCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGC ACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGAT GAAGAAGTTC ATC C C AGGAC GCTGC C AC AC CTAC GAAGGC GAC AAAG

AGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATT CCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGA TCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGC AGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTT GCCAGCAAGATCCAGGGCCAGGTGGACAAG [Xbal] [Xhol]

ATCAAGGGGGCCGGTGGTGACTCTAGA-[Epitope tag site]-CTCGAG

Epitope tag sequences:

Myc:

GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG TGA

Flag:

GAC TAC AAA GAC CAT GAC GGT GAT TAT AAA GAT CAT GAC ATC GAC TAC AAG GAT GAC GAT GAC AAG TGA

His:

CAT CAT CAC CAT CAC CAC TGA HA:

TAT CCG TAT GAT GTG CCG GAT TAT GCG TGA

Kt3:

AAG CCT CCA ACA CCT CCA CCT GAG CCT GAG ACC TGA AU1:

GAC ACC TAC AGA TAC ATC TGA

AcV5:

AGC TGG AAG GAC GCC AGC GGC TGG AGC TGA

V5:

GGC AAG CCT ATC CCT AAC CCT CTG CTG GGC CTG GAC AGC ACC TGA

E2:

AGC AGC ACC AGC AGC GAC TTC AGA GAC AGA TGA

Glu:

TGC GAG GAA GAG GAA TAC ATG CCT ATG GAG TGA Supplementary table SI. Sequence for different epitope tags and antibodies used in this study.

Tag Sequence of oligonucleotides Antibod Com any name 1: sense, 2: antisense 5'-3' y Cat# clone

3x Flag 1 : CT AG AG ACT AC AAAG AC CAT G AC G GT GAT Anti- Sigma-

TATAAAGATCATGACATCGACTACAAGGAT Flag M2 Aldrich

GACGATGACAAGTGAC F1804

2 : T C G AGT C ACTT GT CAT C GT CAT C CTT GT A

GTCGATGTCATGATCTTTATAATCACCGTCA

TGCTGTTTGTAGTCT

6x His 1 : CT AG AC AT CAT C AC CAT C AC C ACT G AC 6xHis Abeam

2:TCGAGTCAGTGGTGATGGTGATGATT tag ab81663

ADl.1.1

0

HA 1 : CTAGATATCCGTATGATGTGCCGGATTAT Hemaggl Abeam

GCGTGAC utinin ab59076

2:TCGAGTCACGCATAATCCGGCACATCATA HA.C5

CGGATAT

AcV5 1:CTAGAAGCTGGAAGGACGCCAGCGGCTG AcV5 Abeam

GAGCTGAC tag ab49581

2:TCGAGTCAGCTCCAGCCGCTGGCGTCCTT AcV5

CCAGCTT

V5 1 : CT AGAGGC AAGC CT ATC C CT AAC C CT CT G V5 tag Abeam

CTGGGCCTGGACAGCACCTGAC SV5-Pkl ab27671 2:TCGAGTCAGGTGCTGTCCAGGCCCAGCAG

AGGGTTAGGGATAGGCTTGCCT

Glu-Glu 1 : CTAGATGCGAGGAAGAGGAATACATGCCT Glu-Glu Abeam

ATG GAGTGAC tag Ab24627

2:TCGAGTCACTCCATAGGCATGTATTCCTC Glu-Glu

TTCCTCGCAT

Myc 1 : CTAGAGAACAAAAACTCATCTCAGAAGAG Anti-c- Sigma- GAT CTGTGAC Myc Aldrich

2 : T C G AGT C AC AG AT C CT CTT CT G AG AT GAG 9E10 M 4439 TTTTTGTTCT

Kt3 1 : CT AGAAAGC CTC C AAC AC CT C C AC CT GAG KT3 tag Abeam

CCT GAGACCTGAC KT3 ab24739

2:TCGAGTCAGGTCTCAGGCTCAGGTGGAGG

TGTTGGAGGCTTT

Aul 1 : CT AG AG AC AC CTAC AGAT AC ATCT GAC Covance

2:TCGAGTCAGATGTATCTGTAGGTGTCT MMS-

130P

E2 1:CTAGAAGCAGCACCAGCAGCGACTTCAGA E2 tag Abeam

GAC AGATGAC 5E11 ab977

2:TCGAGTCATCTGTCTCTGAAGTCGCTGCT

GGTGCTGCTT

Primer sequences for cloning TREs

Oct4

5'-GATCC ATGCAAATAA ATGCAAATAA ATGCAAATAA ATGCAAATAA ATGCAAATAA ATATA G-3'

3'-G TACGTTTATT TACGTTTATT TACGTTTATT TACGTTTATT TACGTTTATT TATAT CGATC-5'

61bp

Sox2

5'-GATCC AACAAAGAGT AACAAAGAGT AACAAAGAGT AACAAAGAGT AACAAAGAGT ATATA G-3'

3'-G TTGTTTCTCA TTGTTTCTCA TTGTTTCTCA TTGTTTCTCA TTGTTTCTCA TATAT CGATC-5'

61bp c-Myc 5'-GATCC CACGTG CACGTG CACGTG CACGTG CACGTG ATATA G-3'

3'-G GTGCAC GTGCAC GTGCAC GTGCAC GTGCAC TATAT CGATC-5'

41bp Klf4

5'-GATCC AGGGTGTGGCC AGGGTGTGGCC AGGGTGTGGCC

AGGGTGTGGCC AGGGTGTGGCC ATATA G-3'

3'-G TCCCACACCGG TCCCACACCGG TCCCACACCGG

TCCCACACCGG TCCCACACCGG TATAT CGATC-5'

66bp

E2F/DP1

5'-GATCC TTTCGCGGGAAA TTTCGCGGGAAA TTTCGCGGGAAA

TTTCGCGGGAAA TTTCGCGGGAAA ATATA G-3'

3'-G AAAGCGCCCTTT AAAGCGCCCTTT AAAGCGCCCTTT

AAAGCGCCCTTT AAAGCGCCCTTT TATAT CGATC-5'

71bp p53

5'-GATCC AGACATGTCCAGACATGTCC GAACATGTCCCAACATGTTGT AGACATGTCCAGACATGTCC GAACATGTCCCAACATGTTGT ATATA G-3'

3'-G TCTGTACAGGTCTGTACAGG CTTGTACAGGGTTGTACAACA TCTGTACAGGTCTGTACAGG CTTGTACAGGGTTGTACAACA TATAT CGATC-5'

93bp

Hifla

5'-GATCC TACGTGCT TACGTGCT TACGTGCT TACGTGCT TACGTGCT ATATA G-3' 3'-G ATGCACGA ATGCACGA ATGCACGA ATGCACGA ATGCACGA TATAT CGATC-5'

51bp Nanog

5'-GATCC ACCCTTCGCCGATTAAGTACTTAA

ACCCTTCGCCGATTAAGTACTTAA ACCCTTCGCCGATTAAGTACTTAA ATATA G-3'

3'-G TGGGAAGCGGCTAATTCATGAATT

TGGGAAGCGGCTAATTCATGAATT TGGGAAGCGGCTAATTCATGAATT TATAT CGATC-5'

83bp

API

5'-GATCC TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG TGAGTCAG ATATA G-3'

3'-G ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC ACTCAGTC TATAT CGATC-5'

91bp

Elkl/SRF

5'-GATCC GGATGTCCATATTAGGA GGATGTCCATATTAGGA

GGATGTCCATATTAGGA GGATGTCCATATTAGGA GGATGTCCATATTAGGA ATATA G-3'

3'-G CCTACAGGTATAATCCT CCTACAGGTATAATCCT

CCTACAGGTATAATCCT CCTACAGGTATAATCCT CCTACAGGTATAATCCT TATAT CGATC-5'

96bp Stat3

5'-GATCC GT C G AC ATTTC C C GT AAATC GT C G A

GTC GAC ATTTC C C GT AAATC GTC GA GTC GAC ATTTC C C GT AAATC GTC GA ATATA G-3'

3'-G

CAGCTGTAAAGGGCATTTAGCAGCTCAGCTGTAAAGGGCATTTAGCAGCT

CAGCTGTAAAGGGCATTTAGCAGCT TATAT CGATC-5'

86bp SPl

5'-GATCC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC GGGGCGGGGC ATATA

G-3'

3'-G CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG CCCCGCCCCG TATAT CGATC-5'

91bp

Creb

5'-GATCC TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA TGACGTCA ATATA G-3'

3'-G ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT ACTGCAGT TATAT CGATC-5'

91bp

NF-kB (5NF, Chris Badr et al. Mol. Imaging, 2009)

5'-GATCTT GGGGACTTTCCGCT GGGGACTTTCCGCT

GGGGACTTTCCGCT GGGGACTTTCCGCT GGGGACTTTCCGCA-3' 3'-AA CCCCTGAAAGGCGA CCCCTGAAAGGCGA CCCCTGAAAGGCGA CCCCTGAAAGGCGA CCCCTGAAAGGCGTGATC-5'

Restriction sites

BamHI

G I GATC C C CTAG I G

Nhel

G I CTAG C C GATC I G

HIV-1 subtype E TATA

ATATA

Example

1. The CSCW-Gluc-IRES-CFP lentiviral vector was used to demonstrate the multiplex method using Gaussia luciferase. This lentiviral vector co- expresses the Glue bioluminescent reporter and the cerulean fluorescent protein (CFP) control. The internal ribosomal entry site (IRES) allows co- expression of both proteins using the same cytomegalovirus (CMV) promoter. The vector is designated CSCW-Gluc-CFP in this protocol (Figure 3).

1. Construct the Glucmodified gene by amplifying the Glucparental gene from lentiviral vector CSCW-Gluc-CFP (see figure 4) making a PCR reaction mix of 20 μΐ using primers that exclude the STOP codon and add the unique restriction site Xbal downstream. Use a high fidelity proof reading DNA polymerase and follow the manufacturer's guidelines to amplify the

Glucmodified gene (633 bp) in thermal cycler by 1: denature the plasmid DNA for 2 minutes at 95°C. Then 2: denature for 30 seconds at 95°C, 3: anneal primers for 30 seconds at 62°C and 4: elongate DNA for 1 minute at 72°C and cycle sequence 2, 3, 4 for 35 times. Allow 5: final elongation of DNA for 2 minutes at 72°C. 2. To form sticky ends to the Glucmodified gene in order to clone it back into the CSCW lentiviral backbone to replace the Glucparental gene, restrict the Glucmodified gene with restriction enzyme Nhel upstream, and restriction enzyme Xhol downstream. Restrict the total volume of the PCR reaction from (1.) by making a double digestion using high fidelity enzymes (see note 3), according to the manufacturer's guidelines in a total volume of 50 μΐ. Incubate the restriction mix for 2 hours at 37°C to ensure complete restriction. Also, create the CSCW lentiviral backbone by restricting 2 μg of the parental CSCW-Gluc-CFP lentiviral vector with Nhel and Xhol as described to restrict out the Glucparental gene.

3. Isolate and purify the Glucmodified gene from (2.) and the CSCW lentiviral backbone from (2.) on a 0.5% (w/v) agarose DNA gel. Add DNA loading buffer (final concentration is lx DNA loading buffer) to the

Glucmodified gene and CSCW lentiviral backbone restriction mixtures from (2.) and load both the samples into a separate gel well. Also load a separate well with a DNA molecular weight marker to identify the product and confirm product sizes. Run the gel in 1 x TAE buffer at 100 volts until the Glucparental gene (718 bp) has separated properly from the CSCW lentiviral backbone (9408 bp).

4. Extract the Glucmodified gene construct (570 bp) and the CSCW lentiviral backbone (9408 bp) using a DNA gel extraction kit according to manufacturer's protocol. After elution with 50 μΐ elution buffer, heat both the DNA extractions in an open tube to 60°C for 5 minutes to completely evaporate any residual ethanol.

5. To clone the Glucmodified gene into the CSCW lentiviral backbone, ligate 100 ng CSCW lentiviral backbone from (4.) with 57 ng Glucmodified gene from (4.) ((Glucmodified gene):(CSCW lentiviral backbone) is 10:1 molar ratio, see note 8) using a T4 DNA ligase according to the manufacturer's guidelines. Use a total reaction volume of 20 μΐ. 6. Transform the resulting ligation product CSCW-Glucmodified-CFP lentiviral vector in ultracompetent bacterial cells for ligated DNA according to the manufacturer's guidelines in order to make bacterial clones for CSCW- Glucmodified-CFP lentiviral vector production. Plate the bacterial cells on a LB + ampicillin (50 μg/ml) bacteria selection and propagation plate and grow the bacterial colonies overnight at 37°C.

7. Amplify and isolate the CSCW-Glucmodified-CFP lentiviral vector using a DNA plasmid kit following the manufacturer's guidelines.

8. Construct epitope tag inserts by annealing the corresponding epitope tag sense and antisense oligonucleotides (see Supplementary Table 1). Mix 50 μΐ of H20 with 10 μΐ of the lOx annealing buffer, 20 μΐ of the sense

oligonucleotide (100 μΜ) and 20 μΐ of the antisense oligonucleotide (100 μΜ). Heat the annealing mixture in a heat block for 10 minutes at 65°C, then take out the metal heat block insert to very slowly cool down the annealing mixture to room temperature.

9. Restrict 2 μg of the CSCW-Glucmodified-CFP using restriction enzymes Xbal and Xhol to open up the CSCW lentiviral backbone in order to insert the epitope tag from (8.). Make a 20 μΐ double digestion using high fidelity restriction enzymes and follow manufacturer's guidelines.

10. Isolate and purify the CSCW lentiviral backbone as described in (3.) and clone the epitope tag insert into the CSCW lentiviral backbone as described in (5.). Use a molar ratio (epitope insert):(CSCW lentiviral vector, see note 8) of 10: 1 and a total volume of 20 μΐ. Transform the resulting CSCW- GlucTag-CFP lentiviral vector hgation product as described in (6.) and amplify the vector as described in (7.).

11. HEK293T and U87 cells are cultured in DMEM complete culture medium at 37°C and 5% C02. The cells are diluted 1/10 when the culture vessel is ~90% confluent. To dilute, aspirate the culture medium and was attached cells with PBS. Shake culture vessel gently and aspirate PBS. Detach the cells by adding lx Trypsin + EDTA, enough to just cover the surface of the culture vessel. Incubate the cells 5 minutes at 37°C for 5 minutes. Resuspend the cells in DMEM complete culture medium (>5x the Trypsin + EDTA volume) and aspirate 9/10 of the total volume to discard or collect the suspended cells. Add DMEM complete culture medium to the 1/10 leftover cell suspension up to the final culture volume. Make sure the surface of the culture vessel is covered with ~0.5 cm culture medium. Use room temperature reagents.

12. Produce the lentiviral particles as described in (Ref. Dull et al.). Transiently transfect 5.5 x 106 HEK293T cells in a 10 cm2 culture dish with 3 μg pMD2.G envelope plasmid, 5 μg pMDLg/pRRE and 2.5 μg pRSV/Rev packaging plasmids and 10 μg CSCW-GlucTag-CFP lentiviral vector using the calcium phosphate transfection method (Ref, see note 9). Harvest the virus- containing medium and spin at 1000 x G for 5 minutes to remove residual cells and debris. Aliquote and store the virus-containing medium at 4°C for use within a week or at -80°C for longer periods of time.

13. Make U87 cell lines (see note 10) stably expressing GlucTag-CFP by lentiviral transduction as described in (Dull et al.). Transduce 2 x 105 U87 cells in a 6 well plate with CSCW-GlucTag-CFP lentivirus (MOI of 5, see note 11) overnight. The following day, replace the virus-containing medium with 2 ml fresh DMEM complete culture medium. Expand and culture the cells as described in (11.). Transduction efficiency can be determined by fluorescence microscopy of CFP (455-480 nm).

3.2 GlucTag-CFP multiplex assay application

GlucTag immunobinding assay in vitro

1. Plate U87-Gluc6x Tag-CFP cells (see note 10) in a 6-well plate in

DMEM complete culture medium and culture the cells in 37°C and 5% C02. For a triplo experiment, plate 3 wells of a 6-well plate. 2. At predetermined time points, collect 180 μΐ (30 μΐ x 6 Tags) of the Gluc6x Tag conditioned culture medium of the 3 wells and store the culture medium at 4°C (see figure 4).

3. After the final time point collection, Wash 18 wells (6 Tags in triplo) per time point of a goat anti-mouse IgG coated 96-well microplate 3 x with 200 μΐ wash buffer (see note 5).

4. Per timepoint, prepare 150 μΐ wash buffer with mouse anti-Tag monoclonal IgG (10 μg/ml, see Table 1, see note 6) for all 6 Tags. Coat the wells with 50 μΐ anti-Tag monoclonal mixture per well (see figure 4) and incubate for 1 hour at 4°C spinning at 500 x G followed by 1 hour of incubation at room temperature on a microplate shaker at 65-75 RPM (to create a gentle swirl in the wells).

5. Then, aspirate anti-Tag monoclonal mixture and the wash the wells 3 x with 200 μΐ of block buffer for 5 minutes on a microplate shaker at 65-75 RPM (to create a gentle swirl in the wells).

6. To bind the GlucTag from the conditioned culture medium, add 30 μΐ of the Gluc6x Tag conditioned culture medium to a mouse anti-tag coated well and incubate at room temperature for 2 hours on a microplate shaker with 65- 75 RPM (to create a gentle swirl in the wells).

7. After this, aspirate the culture medium and wash the wells 5 x for 5 minutes with 200 μΐ wash buffer on a microplate shaker at 65-75 RPM

(creating a gentle swirl in the wells).

8. To measure the bound GlucTag in the mouse anti-Tag coated wells, aspirate the wash buffer completely and just before measurement add 50 μΐ coelenterazine Glue substrate per well using a multichannel pipette.

Immediately insert the plate in the microplate luminometer (see note 11), shake the plate briefly and read out the wells for 0.1 second per well. GlucTag Immunostaining in vitro

1. Plate 105 of the U87-Gluctag cell lines (see note 10) in a 24 well plate overnight in 500 μΐ DMEM complete culture medium at 37°C and 5% C02.

2. The next day, wash the cells with 200 μΐ PBS and fix with 100 μΐ

3.7% formaldehyde fixative solution for 20 minutes.

3. Then, wash and block the cells 3 x 5 minutes with 200 μΐ block buffer and permeabilize the cells with 100 μΐ permeabilization buffer for 15 minutes.

4. Next, wash and block the cells 2 x 5 minutes with 200 μΐ block buffer.

5. Add 100 μΐ primary mouse anti-tag IgG (2 μg/ml, see note 6) in block buffer to the cells and incubate for 1 hour at room temperature.

6. Subsequently, wash and block the cells 3 x 5 minutes with 200 μΐ block buffer.

7. Add 100 μΐ secondary goat anti-mouse-HRP antibody (5 μg/ml, see note 12) in block buffer to the cells and incubate for 1 hour at room

temperature.

8. Then, wash the cells 3 x for 5 minutes with 200 μΐ block buffer and stain the cells with the DAB reagent set according to the manufacturers guidelines. Analyse the immunostaining using light microscopy (see figure 5). GlucTag immunobinding assay in vivo

1. Culture and collect the U87-GlucTag-CFP cells as described before.

Collect 5 x 105 U87-GlucTag-CFP cells per orthotopical injection in athymic nude-foxnlnu mouse. For a triplo experiment inject 3 mice.

2. 30 minutes before surgery of the mouse, subcutaneously administer

Temgesic (Buprenorfinehydrochloride, 0.1 mg/kg) in PBS analgesia and anesthetize the mouse using an isoflurane anaesthesia and fix the mouse in a small animal stereotaxic frame.

3. Incise the head skin with a surgical scalpel and locate the injection site x = 2 mm and y = 0.5 mm from bregma. Drill a hole in the skull and slowly inject the U87-GlucTag-CFP cell suspension into the brain. Then, cover up the skull with the head skin and stitch the wound.

4. At predetermined time points, collect 100 μΐ of the GlucTag conditioned mouse blood of all the mice using a capillary collection and sample container containing EDTA to prevent blood clotting and store the blood at 4°C (see note 7).

5. After the final blood sample collection, dilute the blood 1: 1 with blood dilution buffer to increase the sample volume to 180 μΐ.

6. Wash 18 wells (6 Tags in triplo) per time point of a goat anti-mouse IgG coated 96-well microplate (see note 5) 3 x with 200 μΐ wash buffer.

7. Per timepoint, prepare 150 μΐ wash buffer with mouse anti-Tag monoclonal IgG (10 μg/ml, see Table 1) for all 6 Tags (see note 13). Coat the wells with 50 μΐ anti-Tag monoclonal mixture per well and incubate for 1 hour at 4°C spinning at 500 x G followed by 1 hour of incubation at room

temperature.

8. Then, aspirate anti-Tag monoclonal mixture and the wash the wells 3 x with 200 μΐ of block buffer for 5 minutes on a microplate shaker at 65-75 RPM (to create a gentle swirl in the well).

9. To bind the GlucTag from the conditioned mouse blood, add 30 μΐ of the diluted Gluc6x Tag blood samples to a mouse anti-tag coated well and incubate at room temperature for 2 hours on a microplate shaker with 65-75 RPM (to create a gentle swirl in the well).

10. After this, aspirate the diluted Gluc6x Tag blood samples and wash the wells 5 x for 5 minutes with 200 μΐ wash buffer on a microplate shaker at 65-75 RPM (creating a gentle swirl).

11. To measure the bound GlucTag in the mouse anti-Tag coated wells, aspirate the wash buffer completely and just before measurement add 50 μΐ coelenterazine Glue substrate per well using a multichannel pipette.

Immediately insert the plate in the microplate luminometer plate reader, shake the plate briefly and read out the wells for 0.1 second per well (see note 11).

GlucTag Immunostaining in vivo

1. After the in vivo GlucTag assay, collect and fix the mouse brains containing the tumor in 3.7% formaldehyde for at least 48 hours. Then, dehydrate and embed the tissue samples in paraffin.

2. Using a microtome, section the paraffin embedded tissue samples in

5 μιη slices and mount the slices on glass slides.

3. Deparaffinise the tissue slices in xylene and rehydrate in a series of

100% ethanol, 96% ethanol and 75% ethanol.

4. Block the endogenous peroxidase with fresh 0.3% H202 in methanol for 30 minutes at room temperature and rinse the slices with H20.

5. Perform antigen retrieval by cooking the slices in citrate buffer (pH 6) using a microwave. Cook the samples 5 minutes at 100% power and 10 minutes at 50% power. Cool the slices down to room temperature in 20 minutes and rinse the slices 3 x 5 minutes with PBS.

6. Incubate the slices with mouse anti-Tag monoclonal IgG (final concentration is 2 μg/ml, see note 6) diluted in in antibody diluent for 1 hour at room temperature. After IgG incubation, rinse the slices 3 x 5 minutes with PBS.

7. Incubate the slices with anti-mouse-HRP (1 in 200) diluted in antibody diluent for 30 minutes at room temperature and rinse the slices 3 x 5 minutes in PBS.

8. Incubate the slices with DAB for 5 to 10 minutes and rinse the slices with H20.

9. Counterstain the cell nuclei of the tissue slices with haematoxyline for 30 to 60 seconds and rinse the slices with PBS. 10. Dehydrate the slices in the ethanol series of 75% ethanol, 96% ethanol and 100% ethanol and incubate the slices in xylene for 5 minutes. Let the tissue slices dry and analyse them using a light microscope (see figure 5). 4 Notes- Preferred embodiments

1. In the assay described here, we use the CSCW-Gluc-IRES-CFP lentiviral vector DNA as a template to amplify the Glue reporter gene and to religate our GlucTag reporter constructs in. When using other template vectors or reporters, it is necessary do redesign primers, restriction sites and oligonucleotide sequences and PCR programs should be optimised in order to fit the alternative vector DNA or reporter gene.

2. Preferably, a high fidelity proof reading DNA polymerase is used for the amplification of the Glue reporter gene. This minimizes optimization and the risk of copy errors due to incorrect basep airing.

3. Preferably, high fidelity restriction enzymes for restriction of the DNA vectors are used. This increases restriction efficiency and user simplicity since most high fidelity enzymes are optimised in the same restriction reaction buffer. Also, most high fidelity restriction enzymes are optimised to have less star activity, increasing ligation efficiency in later steps of the protocol.

4. There are less hazardous alternatives available for ethidium bromide, such as SYBR safe DNA gel stain (Life Technologies). Besides being less hazardous, these stains can also be used to visualize DNA with non-UV- light, decreasing damage of the DNA due to UV exposure.

5. For luciferase assays, preferable, white microplates are used to prevent crossover detection of photons between wells. The white goat anti- mouse IgG coated microplates (Thermo Scientific) are pre-coated to improve assay stability but it is also possible to manually coat microplates (e.g. ELISA microplates).

6. The antibodies we used are all mouse monoclonal IgG antibodies. The coated microplates used (see note 5) are optimised to use with mouse IgG antibodies. Using monoclonal antibodies over polyclonal antibodies improves assay stability.

7. The capillary collection and sample container (Sarstedt) we used combines a capillary blood collector with an EDTA coated sample container. This enables fast and easy sample collection and storage. If you collect mouse blood using other methods, make sure you add EDTA to the blood sample to prevent clotting.

8. To quantify the concentration of the DNA gel extracts, we do not recommend the use of spectrophotometry (Nanodrop) since the concentration will usually be low (<100 ng/μΐ). We advise to quantify the concentrations of the DNA gel extracts by loading a small amount of DNA sample on a gel and quantify the resulting bands by comparing to the molecular marker. We used ImageJ to quantify the DNA sample concentrations and calculate the ligation conditions.

9. Generally, higher virus titers are obtained using other methods of transfection of the plasmids and vectors. Using Lipofectamine 2000 (Life Technologies) or Fugene (Promega) can be beneficial for virus production titres.

10. It is possible to transduce other cell lines of your interest but not all cell lines are equally resistant to lentiviral transduction and therefore the transduction conditions need to be optimized for every cell line. To increase transduction efficiency, it might be beneficial to culture the cells with polybrene (2-10 μg/ml) before transduction. Also using polybrene requires optimization, depending on the cell line of use. Expression of the reporters is also cell line dependent since the CMV promoter is not equally active in all cell lines and therefore also the promoter might be an issue for optimization.

11. Since the Glue photon signal is degrading over time (~10% per minute), the most stable method of measuring would be to measure Glue signal directly after coelenterazine substrate addition. A plate reader with a substrate injector would be optimal but it is also an option to use a multichannel pipette. Measuring a 96-well microplate for 0.1 second per well would take about 10 seconds, so the time difference as a result of measuring the first well and the last well would be well within 10% deviation as a result of the Glue signal degradation.

12. For this assay we used goat anti-mouse secondary Ig. It would also be possible to use other anti-mouse Ig antibodies.

13. As a negative control for the immunobinding assays and the stainings it is possible to use the CSCW-Glucctrl-CFP vector. This vector contains the Gaussia luciferase not fused with an epitope tag_..

Claims

Claims

1. A method for monitoring an effect on a biological system comprising providing said biological system with at least two reporters,

wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to a reporter;

exposing said biological system to a biological condition; and

determining the level of said reporters.

2. The method of claim 1, wherein the marker is Gaussia luciferase.

3. The method of any of the preceding claims, wherein at least three reporters are provided to said biological system.

4. The method of any of the preceding claims, wherein the biological system is one or more cells.

5. The method of claim 4, wherein the cells are tumor cells.

6. The method of any of the proceeding claims, wherein the marker from each reporter is the same.

7. The method of claim 6, further comprising distinguishing said reporter based on the epitope tag.

8. The method of any of the proceeding claims, wherein the marker is secreted.

9. The method of any of the proceeding claims, wherein said reporters are provided to said biological system in vitro.

10. The method of claim 9, wherein said biological system is implanted into an animal.

11. The method of claim 9, where said reporter level is determined in a bodily fluid, preferably in the blood, of said animal.

12. A reporter system comprising at least two reporters,

wherein each reporter comprises a marker, preferably a luminescent marker, linked to an epitope tag, and each epitope tag is unique to each reporter.

13. The reporter system of claim 10, wherein the marker is Gaussia luciferase.

14. The reporter system of claims 12 or 13, comprising at least three reporters.

15. The reporter system of any one of claims 12-14, wherein each reporter comprises a different transcription response element or a different promoter sequence.

16. The reporter system of any one of claims 12-15, wherein each reporter comprises a different protease cleavage site.

17. The reporter system of any one of claims 12-16, wherein each reporter comprises a different miRNA binding sequence or a different RNA splicing sequence.

18. The reporter system of any one of claims 12-17 or the method of any one of claims 1-11,, wherein the epitope tag is selected from Flag, His, HA, AcV5, V5, Glu, Myc, Kt3, Aul, and E2.

19. The reporter system or method of claim 15, wherein said epitope tag is selected from Flag, His, HA, AcV5, V5, and Glu.

20. The reporter system of any one of claims 12-19, wherein said reporters are provided in one or more vectors.

21. Nucleic acid encoding the reporter system of any one of claims 12-20.

22. A cell comprising the nucleic acid of claim 21.